Method for producing nanocelluloses from a cellulose substrate

ABSTRACT

Disclosed is a method for producing nanocelluloses from a cellulose substrate including cellulose fibers, the method including the following sequence of steps: —a step of enzymatic treatment of the cellulose substrate, by bringing same into contact with at least one cleaving enzyme, then—a step of mechanical treatment of the cellulose substrate subjected to the step of enzymatic treatment, in order to delaminate the cellulose fibres and obtain the nanocelluloses. The at least one cleaving enzyme is chosen from the enzymes belonging to the family of lytic polysaccharide monooxygenases (LPMOs) capable of achieving cleavage in the presence of an electron donor. Also disclosed are the nanocelluloses obtained according to the method.

TECHNICAL FIELD TO WHICH THE INVENTION RELATES

The present invention relates, generally, to the field ofnanocelluloses, and more particularly the processes for producing thesenanocelluloses from a cellulose-based substrate.

TECHNICAL BACKGROUND

Cellulose is one of the most important natural polymers, a virtuallyinexhaustible raw material, and an important source of materials thatare sustainable on the industrial scale.

To date, various forms of cellulose have been identified with a size ofabout one nanometer, denoted under the generic name of “nanocelluloses”.

The properties of these nanocelluloses, in particular their mechanicalproperties, their ability to form films and their viscosity, gives thema major advantage in numerous industrial fields.

Nanocelluloses are thus used, for example, as a dispersant orstabilizing additive in the papermaking, pharmaceutical, cosmetics orfood-processing industries. They are also part of the composition ofpaints and varnishes.

Nanocelluloses are also used in many devices that require a control ofnanometric porosity, owing to their high specific surface area.

Finally, many nanocomposite materials based on nanocelluloses arecurrently being developed. This is because the notable mechanicalproperties of nanocelluloses, their dispersion on the nanometric scaleand also their hydrophilic nature, give them excellent gas-barrierproperties. These characteristics create in particular a considerableadvantage for the production of barrier packagings.

On the basis of their sizes, functions and preparation methods, whichthemselves depend mainly on the cellulose-based source and on thetreatment conditions, nanocelluloses can be classified mainly in twofamilies: cellulose fibrils and cellulose nanocrystals.

Cellulose nanocrystals (also known as NCCs for “nanocrystallinecelluloses”) are generally obtained by hydrolysis with a strong acidunder strictly controlled temperature, time and stirring conditions.Such a treatment makes it possible to attack the amorphous regions ofthe fibers while at the same time leaving the more resistant crystallineregions intact. The suspension obtained is then washed bycentrifugations and successive dialyses in distilled water. The NCCsmost conventionally obtained have a length of a few tens of nanometersto approximately 1 μm (in particular from 40 nm to 1 μm and preferablyfrom 40 nm to 500 nm), and a diameter ranging from 5 to 70 nm,preferably less than 15 nm (typically from 5 to 10 nm).

Cellulose fibrils, commonly denoted cellulose microfibrils (alsoreferred to as MFC for “microfibrilated cellulose”) or cellulosenanofibrils (NFC for “nanofibrilated cellulose”) are typically isolatedfrom cellulose-based materials derived from biomass, by mechanicalprocesses which make it possible to delaminate the cellulose fibers andto release the cellulose fibrils.

For example, document U.S. Pat. No. 4,483,743 describes a process forproducing microfibrilated cellulose, which involves passing a liquidsuspension of cellulose through a high-pressure Gaulin homogenizer.Repeated passes of the cellulose suspension make it possible to obtainmicrofibrils which typically have a width ranging from 25 to 100 nm anda much longer length.

In general, the mechanical processes for obtaining cellulose fibrilshave the drawback of consuming large amounts of energy. By way ofexample, it has been evaluated that the use of a homogenizer causes anenergy consumption of about 70 000 kWh/t. This high energy consumption,and consequently the high costs of producing nanocelluloses, thereforeremain a considerable impediment to their industrial development.

Various cellulose fiber pretreatment strategies have thus been developedin order to reduce the energy consumption required for their mechanicaldelamination.

A first pretreatment strategy, described for example in application WO2007/091942, consists in pretreating the cellulose fibers withcellulases so as to destroy the fiber before the application of themechanical treatment by homogenization.

However, this enzymatic pretreatment is extremely changeable dependingon the condition of the fiber and in particular depending on the priorthermochemical history of the fiber.

Furthermore, the quality of the nanocelluloses obtained (in particularthe dispersion state and especially the lateral size of the nanofibrilswhich conditions the wear properties and the energy yields) are veryvariable.

A second pretreatment strategy is based on a chemical step of oxidationof the cellulose fibers (for example, Saito et al., Biomacromolecules,Vol. 8, No. 8, 2007, pp. 2485-2491).

Typically, the fibers are oxidized with an oxidizing agent such assodium hypochlorite catalyzed by the2,2,6,6-tetramethylpiperidine-1-oxyl (“TEMPO”) radical, beforeundergoing the abovementioned mechanical treatment.

The oxidative treatment converts the primary alcohol function in the 0₆position of the glucose unit of the cellulose into a carboxylatefunction, which results in the introduction of charges at the surface ofthe cellulose fibers. These charges create electrostatic repulsionswhich facilitate the delamination and which increase its efficiency.

However, the removal of the reaction products results in large amountsof highly polluted effluents. In addition, reactant residues persist inthe final product and continue to react, altering, in the end, theproperties of the nanocelluloses.

Thus, despite the new pretreatment strategies developed, the costs ofnanocellulose production remain high, the yields uncertain, and thequality and the properties are variable.

It thus remains necessary to provide new processes for obtainingnanocelluloses, with a lower energy consumption and which are simple andreproducible, according to a route that is not toxic or has lowtoxicity.

SUBJECT OF THE INVENTION

In order to overcome the abovementioned drawbacks of the prior art, thepresent invention provides a process for producing nanocelluloses whichis based on a step of pretreating cellulose fibers with at least oneenzyme belonging to the family of lytic polysaccharide monooxygenases,commonly denoted “LPMOs”.

More particularly, according to the invention, a process is provided forproducing nanocelluloses from a cellulose-based substrate comprisingcellulose fibers,

said process comprising the following successive steps:

-   -   at least one step of enzymatic treatment of said cellulose-based        substrate, by bringing it into contact with at least one        cleavage enzyme, then    -   at least one step of mechanical treatment of said        cellulose-based substrate subjected to said at least one step of        enzymatic treatment, in order to delaminate the cellulose fibers        and to obtain said nanocelluloses,

characterized in that said at least one cleavage enzyme is chosen fromthe enzymes belonging to LPMO family.

Typically, LPMOs are capable of performing an oxidative cleavage ofcellulose fibers, advantageously of the glucose rings of cellulosefibers, in the presence of an electron donor.

Without being limited by any theory, the action of LPMOs facilitates theproduction of nanocellulose through two actions:

-   -   the cleavage of the cellulose-based chains causes fragilities        within the fibers, facilitating the mechanical delamination,    -   the formation of oxidation products makes it possible to        introduce charged chemical functions on the surface of the        fibers, inducing electrostatic repulsions.

Again without being limited by any theory, the consequence of thesecombined structural modifications is to promote the separation of thefibers until nanometric dispersion is obtained and to formnanocelluloses (fibrils or nanocrystals) which have new functionalities(degree of charges, chemical functions not currently available).

Other nonlimiting and advantageous characteristics of the productionprocess in accordance with the invention, taken individually oraccording to all the technically possible combinations, are alsodescribed hereinafter and also in the detailed description of theinvention.

The electron donor can be chosen from ascorbate, gallate, catechol,reduced glutathione, lignin fragments and fungal carbohydratedehydrogenases (in particular glucose dehydrogenases and cellobiosedehydrogenases).

Preferably, the LPMOs are chosen from enzymes capable of carrying out acleavage of the cellulose by oxidation of at least one of the carbonatoms in position(s) C₁, C₄ and C₆ of the glucose ring. More preferably,the LPMOs are chosen from enzymes capable of carrying out a cleavage ofthe cellulose by oxidation of at least one of the carbon atoms inposition(s) C₁ and/or C₄, optionally in combination with C₆, of theglucose ring.

The LPMOs can be chosen from the families of fungal enzymes AA9(formerly known as GH61) and of bacterial enzymes AA10 (formerly knownas CBM33) of the CAZy classification (www.cazy.org). In particular, theLPMOs can be chosen from the LPMOs derived from Podospora anserina andpreferably from PaLPMO9A (Genbank CAP68375), PaLPMO9B (GenbankCAP73254), PaLPMO9D (Genbank CAP66744), PaLPMO9E (Genbank CAP67740),PaLPMO9F (Genbank CAP71839), PaLPMO9G (Genbank CAP73072), and PaLPMO9H(Genbank CAP61476).

According to the embodiments of the invention, the cellulose-basedsubstrate is obtained from wood, from a cellulose-rich fibrous plant,from beetroot, from citrus fruits, from annual straw plants, from marineanimals, from algae, from fungi or from bacteria.

Preferably, the cellulose-based substrate is chosen from chemicalpapermaking pulps, preferably chemical wood papermaking pulps, morepreferably at least one of the following papermaking pulps:

-   -   bleached pulps,    -   semi-bleached pulps,    -   raw pulps,    -   bisulfite pulps,    -   sulfate pulps,    -   sodium hydroxide pulps,    -   kraft pulps.

Said at least one step of mechanical treatment generally comprises atleast one of the following mechanical treatments:

-   -   a homogenization treatment,    -   a microfluidization treatment,    -   an abrasion treatment,    -   a cryomilling treatment.

The process can also comprise a post-treatment step, for example an acidtreatment, an enzymatic treatment, an oxidation, an acetylation, asilylation, or else a derivatization of certain chemical groups borne bythe nanocelluloses.

The invention also relates to the nanocelluloses obtained by carryingout the process of the invention.

Typically, the nanocelluloses obtained consist of cellulose nanofibrilsand/or of cellulose nanocrystals.

Preferably, the nanocelluloses comprise glucose rings of which at leastone carbon atom is oxidized in position(s) C₁ and/or C₄, or even also inposition C₆.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Appearance of kraft fibers of cellulose that have been treatedwith the PaLPMO9H enzyme according to various enzyme/substrate ratiosand subjected to a weak mechanical treatment with ahomogenizer-disperser of the Ultra-Turrax type and an ultrasonictreatment. Optical microscopy images of the fibers not treated (control)with the enzyme (A) or treated with enzyme/substrate ratios of 1:50 (B);1:100 (0); and 1:500 (D).

FIG. 2: Appearance of kraft fibers of cellulose that have been treatedwith the PaLPMO9H enzyme at an enzyme/substrate ratio of 1:50 andsubjected to a weak mechanical treatment with a homogenizer-disperser ofthe Ultra-Turrax type and an ultrasonic treatment. Optical microscopyimages of the control fibers (A) and of the treated fibers (B), andvisualization of the nanofibrils obtained, by transmission electronmicroscopy (C) and atomic force microscopy (D).

FIG. 3: Appearance of kraft fibers of cellulose that have been treatedwith the PaLPMO9H and PaLPMO9E enzymes according to an enzyme/substrateratio of 1:50 and then subjected to a weak mechanical treatment with ahomogenizer-disperser of the Ultra-Turrax type and an ultrasonictreatment. Optical microscopy images of the kraft fibers treated withPaLPMO9H (A) and with PaLPMO9E (B). Visualization by atomic forcemicroscopy of the nanofibrils obtained from the kraft fibers bytreatment with the LPMO enzymes PaLPMO9H (C) and PaLPMO9E (D).

FIG. 4: Appearance of kraft fibers of cellulose that have been subjectedto two successive treatments with the PaLPMO9H enzyme at variousenzyme/substrate ratios and then subjected to a weak mechanicaltreatment with a homogenizer/disperser of the Ultra-Turrax type and anultrasonic treatment. Optical microscopy images of the fibers treatedaccording to the enzyme/substrate ratios of 1:50 (A); 1:100 (B); 1:500(C) and 1:1000 (D).

FIG. 5: Analysis of the AFM photos for the PaLPMO9H enzyme, so as tocharacterize the height profile (FIG. 5A) and the size distribution(FIG. 5B) of the nanocelluloses. Legend to FIG. 5A: example of a heightprofile obtained on the surface, width (x, μm) versus height (y, nm);legend to FIG. 5B: histogram of height distribution with height (x, nm)versus number (y).

DETAILED DESCRIPTION

The description which follows, in combination with the ExperimentalResults given by way of nonlimiting examples, will provide a clearunderstanding of what the invention consists of and how it can becarried out.

General Definitions

The present invention relates to a process for producing nanocelluloses,in particular cellulose fibrils and/or cellulose nanocrystals, from acellulose-based substrate.

The term “cellulose” is intended to mean a linear homopolysaccharidederived from biomass (encompassing organic matter of plant origin, algaeincluded, cellulose of animal origin and also cellulose of bacterialorigin) and consisting of units (or rings) of glucose(D-anhydroglucopyranose—AGU for “anhydro glucose unit”) which are linkedto one another by β-(1-4) glycosidic bonds. The repeat unit is a glucosedimer also known as cellobiose dimer.

AGUs have 3 hydroxyl functions: 2 secondary alcohols (on the carbons inpositions 2 and 3 of the glucose ring) and a primary alcohol (on thecarbon in position 6 of the glucose ring).

These polymers link together via intermolecular bonds of hydrogen bondtype, thus conferring a fibrous structure on the cellulose. Inparticular, the linking of cellobiose dimers forms an elementarycellulose nanofibril (the diameter of which is approximately 5 nm). Thelinking of elementary nanofibrils forms a nanofibril (the diameter ofwhich generally ranges from 50 to 500 nm). The arranging of several ofthese nanofibrils then forms what is generally referred to as acellulose fiber.

The term “nanocelluloses” denotes the various forms of cellulose havinga size of about one nanometer. This encompasses in particular, accordingto the invention, two nanocellulose families: cellulose nanocrystals andcellulose fibrils.

The terms “cellulose fibrils”, “(cellulose) nanofibrils”, “(cellulose)nanofibers”, “nanofibrilated cellulose”, “(cellulose) microfibrils”,“microfibrilated cellulose”, and “cellulose nanofibrils” are synonymous.In the remainder of the present application, the term “cellulosenanofibrils” (NFCs) will be used generically.

Each cellulose nanofibril contains crystalline parts stabilized by asolid network of inter-chain and intra-chain hydrogen bonds. Thesecrystalline regions are separated by amorphous regions.

Elimination of the amorphous parts of cellulose nanofibrils makes itpossible to obtain cellulose nanocrystals (NCCs).

NCCs advantageously comprise at least 50% of crystalline part, morepreferably at least 55% of crystalline part. They generally have adiameter ranging from 5 to 70 nm (preferably less than 15 nm) and alength ranging from 40 nm to approximately 1 μm, preferably ranging from40 nm to 500 nm.

The terms “cellulose nanocrystals”, “nanocrystalline cellulose”,“cellulose whiskers”, “microcrystals” or “nanocrystal cellulose” aresynonymous. In the remainder of the present application, the term“cellulose nanocrystals” (NCCs) will be used generically.

In the case of bacterial cellulose, nanofibrils, or ribbons, ofbacterial cellulose generally have a length of several micrometers and awidth ranging from 30 to 60 nm, in particular from 45 to 55 nm.

Process According to the Invention

The process for producing nanocelluloses, according to the invention,comprises the following successive steps:

-   -   at least one step of enzymatic treatment of a cellulose-based        substrate comprising cellulose fibers, by bringing it into        contact with at least one cleavage enzyme belonging to the        family of lytic polysaccharide monooxygenases (LPMOs), then    -   at least one step of mechanical treatment of said        cellulose-based substrate subjected to said at least one step of        enzymatic treatment, in order to delaminate said cellulose        fibers and to obtain said nanocelluloses.

One or more, and in particular at least two, steps of enzymatictreatment can be carried out according to the process of the invention,prior to said at least one step of mechanical treatment. For example, atleast two steps of enzymatic treatment can be carried out successively,prior to said at least one step of mechanical treatment.

At least one step of enzymatic treatment can also be carried out aftersaid at least one step of mechanical treatment.

When several steps of enzymatic treatment are carried out, the treatmentconditions (time, LPMO(s) chosen, enzyme/cellulose ratio, etc.) can beidentical to or different than one another.

Typically, said at least one step of enzymatic treatment, optionallyfollowed by at least one step of mechanical treatment, can be repeated,as described above, until complete delamination of the cellulose fibersis obtained.

For example, the process of the invention can comprise at least twosuccessive treatment cycles, each treatment cycle comprising at leastone step of enzymatic treatment of the cellulose-based substrate,followed by at least one step of mechanical treatment of said substrate.

Without being limited by any theory, the combination according to theinvention (i) of an enzymatic treatment with at least one LPMO and (ii)of a mechanical delamination treatment makes it possible to obtainnanocelluloses of which the structural characteristics and themechanical properties are entirely different than the nanocellulosesthat exist in the prior art.

Again without being limited by any theory, the process of the inventionmakes it possible to obtain nanocelluloses simply and reproducibly.Preferably, the size and the mechanical properties of thesenanocelluloses are uniform.

Cellulose-Based Substrate

The cellulose-based substrate can be obtained according to the inventionfrom any matter of the biomass (encompassing organic matter of plantorigin, algae included, animal origin or fungal origin) comprisingcellulose-based fibers (that is to say cellulose fibers).

The cellulose-based substrate is advantageously obtained from wood (ofwhich cellulose is the main component), but also from any cellulose-richfibrous plant, for instance cotton, flax, hemp, bamboo, kapok, coconutfibers (coir), ramie, jute, sisal, raffia, papyrus and certain reeds,sugarcane bagasse, beetroot (and in particular beetroot pulp), citrusfruits, corn stalks or sorghum stalks, or else annual straw plants.

The cellulose-based substrates can also be obtained from marine animals(such as tunicates for example), algae (for instance Valonia orCladophora) or bacteria for bacterial cellulose (for instance bacterialstrains of Gluconacetobacter types).

Depending on the applications, cellulose from primary walls, forinstance the parenchyma of fruits (for example beetroots, citrus fruits,etc.), or from secondary walls, for instance wood, will be chosen.

The cellulose-based substrate advantageously consists of acellulose-based material prepared by chemical or mechanical means fromany cellulose-based source as mentioned above (and in particular fromwood).

The cellulose-based substrate is advantageously in the form of asuspension of cellulose fibers in a liquid medium (preferably an aqueousmedium), or of a cellulose pulp.

The cellulose pulps can be conditioned in the “dry” state, that is tosay typically in a state of dryness greater than or equal to 80%, inparticular greater than or equal to 90%. The cellulose pulp cansubsequently be redispersed in an aqueous medium by mechanicaltreatment.

Preferably, the cellulose-based substrate contains at least 90%, inparticular at least 95% and preferably 100% of cellulose fibers.

Preferably, the cellulose-based substrate is suitable for the productionof paper or of a cellulose-based product. The cellulose-based substrateis thus preferably chosen from papermaking pulps (or paper pulp), and inparticular chemical papermaking pulps.

Generally, the cellulose pulp and in particular the papermaking pulp cancontain, in combination with the cellulose fibers, hemicellulose andlignin. Preferably, the cellulose pulp contains less than 10% and inparticular less than 5% of lignin and/or of hemicellulose.

Preferably, the chemical papermaking pulps contain virtuallyexclusively, or even exclusively, cellulose fibers.

The papermaking pulp can be chosen from at least one of the followingpapermaking pulps: bleached pulps, semi-bleached pulps, raw pulps, (rawor bleached) bisulfite pulps, (raw or bleached) sulfate pulps, (raw orbleached) sodium hydroxide pulps and kraft pulps.

It is also possible to use pulps to be dissolved, that have a lowproportion of hemicellulose, preferably less than 10% and in particularless than or equal to 5%.

Preferably, the papermaking pulps used in a process of the invention arewood pulps, in particular chemical papermaking pulps of wood.

Lytic Polysaccharide Monooxydenases—LPMOs

The cellulose-based substrate is thus subjected to at least one step ofpretreatment with at least one cleavage enzyme belonging to the lyticpolysaccharide monooxygenase (LPMO) family.

LPMOs are mononuclear type II copper enzymes. They have commonstructural characteristics, in particular:

-   -   a planar surface with an active site located close to its center        and    -   a highly conserved binding site for a type II copper ion exposed        at the surface of the protein.

The interaction between the LPMO enzyme and the surface of the celluloseoccurs by means of the planar face of the LPMO enzyme and involvesinteractions with polar aromatic residues. The LPMOs that can be usedaccording to the invention are defined by their capacity to catalyze anoxidative cleavage of the cellulose fibers of the cellulose-basedsubstrate, by oxidation of at least one of the carbon atoms in positionsC₁, C₄ and C₆ of a glucose ring of said cellulose fibers.

The principle of the oxidative cleavage carried out by the LPMOsinvolves the activation of a C—H group followed by a dioxygen(O₂)-dependent cleavage, thus producing oligomers that are oxidized onat least one of the carbons in positions C₁, C₄ and C₆.

The LPMO(s) used are capable of catalyzing a cleavage of the cellulosefibers by oxidation of at least one of the carbons chosen from thecarbons in positions C₁ and/or C₄ and/or C₆ of a glucose ring of thecellulose. The oxidative cleavage results in the formation of carboxylgroups at the surface of the cellulose fibers:

-   -   the oxidative cleavage in position C₁ of a glucose ring of a        cellobiose unit leads to the formation of a lactone, which is        spontaneously hydrolyzed to aldonic acid, and    -   the oxidative cleavage in position C₄ of a glucose ring of a        cellobiose unit results in the formation of a ketoaldose.

The oxidation of the alcohol group in position C₆ of a glucose ring of acellobiose unit results in the formation of a carbonyl group.

In some embodiments, the LPMO(s) used catalyze(s) a cleavage of thecellulose fibers by oxidation of at least one of the carbons chosen fromthe carbons in position(s) C₁ and/or C₄ of a glucose ring of thecellulose, optionally in combination with the carbon in position C₆.

LPMOs catalyze the oxidative cleavage of a cellobiose unit in thepresence of an external electron donor.

This electron donor, generally a molecule of low molecular weight, ischosen from ascorbate, reduced glutathione, gallate, catechol, ligninfragments, or else an enzyme of the carbohydrate dehydrogenase family.

Preferably, the carbohydrate dehydrogenases are chosen from fungalenzymes, in particular cellobiose dehydrogenases (CDHs).

CDHs (or cellobiose oxidoreductases—EC 1.1.99.18) catalyze the[cellobiose+electron acceptor<=>cellobiono-1,5-lactone+reduced acceptor]reaction. They are fungal hemoflavoenzymes belonging to theglucose-methanol-choline (GMC) oxidoreductase superfamily. CDHs aremonomeric enzymes bearing two prosthetic groups, a heme group b and aflavin adenine dinucleotide. The flavoprotein domain of CDHs catalyzesthe two-electron oxidation of cellobiose to lactone using an electronacceptor. This electron acceptor can for example be chosen fromdioxygen, quinones and phenoxy radicals or LPMOs.

The activity of a CDH enzyme can be determined according to thereduction of the reagent 2,6-dichlorophenol indophenol (DCPIP) in asodium acetate buffer containing cellobiose (Bey et al., 2011, Microb.Cell Fact. 10:113).

Examples of CDHs that can be used in combination with at least one LPMOenzyme and which also act as an electron donor can be chosen from theCDHs originating from Pycnoporus cinnabarinus, Humicola insolens,Podospora anserina, or Myceliophthora thermophila.

More preferably, an LPMO enzyme for which a cellulolytic activity (thatis to say an activity that catalyzes the oxidative cleavage of thecellulose) has been identified is used. The oxidative cleavage activityof LPMOs on a cellulose-based substrate can be tested in cleavage testsas described in the Example section of the present application.

More specifically, the LPMOs used in the invention are advantageouslychosen from enzymes said to have “auxiliary activity” (AA) according tothe classification established in the CAZy database, relating to enzymesthat are active on carbohydrates (CAZy—Carbohydrate Active enZymedatabase—http://www.cazy.org/—see also Levasseur et al., Biotechnologyfor Biofuels 2013, 6: 41).

More preferably, the step of enzymatic treatment is carried out with atleast one enzyme chosen from the LPMO enzymes of the families referredto as AA9, AA10, AA11 and AA13, according to the classificationestablished in the CAZy database.

The LPMO enzyme according to the invention can contain a carbohydratebinding protein module specific for cellulose of CBM1 type according tothe CAZy classification.

The enzymes listed in the present application are identified by theGenbank reference (identifying a genetic sequence) and the Uniprotreference when the latter is available (identifying a proteinsequence—see table 1). By default, the reference indicated betweenparentheses for each enzyme corresponds to the “Genbank” reference.

Preferably, at least one enzyme of the AA9 family and/or at least oneenzyme of the AA10 family of the CAZy classification is (advantageouslyexclusively) used.

The enzymes of the AA9 family, listed in table 1 hereinafter, are fungalenzymes widely distributed in the genome of most ascomycetes and in somebasidiomycetes (fungi).

Generally, the enzymes of the AA9 family were initially classified inthe family of glycoside hydrolases 61 (GH61) of the CAZy classification.Specific analyses have since shown that the endoglucanase activity ofthe AA9 enzymes is weak, or even nonexistent (Morgenstern I et al.,Briefings in Functional Genomics vol. 3(6P): 471-481).

Preferably, LPMOs of which the endoglucanase activity is not significantor is inexistent are used.

The copper ion of the LPMOs of the AA9 family is bound to the proteinaccording to a hexacoordination model involving at least 2 conservedhistidine residues and water molecules.

The enzymes of the AA9 family catalyze an oxidative cleavage of thecellobiose unit on the carbon in position C₁ and/or C₄, preferably onthe carbon in position C₁ or C₄. Some enzymes (T. aurantiacus TaGH61A(G3XAP7) and Podospora anserina PaGH61 B (B2AVF1)) could catalyze anoxidative cleavage of the cellobiose on a carbon in position C₆.

The LPMOs of the AA9 family that is expressed in fungi generallyexhibits a post-translational modification consisting of a methylationof the N-terminal histidine residue.

Preferably, LPMOs of the AA9 family comprising at least one CBM1 orCBM18 domain (CBM for “carbohydrate binding module”) in the N-terminalposition are used. These enzymes then comprise a planar surface made upof several polar aromatic residues forming a domain of CBM1 or CBM18type.

More preferably, said at least one LPMO of the AA9 family is derivedfrom Podospora anserina and/or from Neurospora crassa.

The enzymes of the AA9 family derived from Podospora anserina aretypically chosen from the group consisting of PaLPMO9A (CAP68375),PaLPMO9B (CAP73254), PaLPMO9D (CAP66744), PaLPMO9E (CAP67740), PaLPMO9F(CAP71839), PaLPMO9G (CAP73072) and PaLPMO9H (CAP61476).

Preferably, the PaLPMO9E (CAP67740) and/or PaLPMO9H (CAP61476) enzymesare used.

The enzymes of the AA9 family derived from Neurospora crassa aretypically chosen from the group consisting of NcLPM09C (EAA36362),NcLPMO9D (EAA32426/CAD21296), NcLPMO9E (EAA26873), NcLPMO9F(EAA26656/CAD70347), NcLPMO9M (EAA33178), NcU00836 (EAA34466), NcU02240(EAA30263) and NcU07760 (EAA29018).

The enzymes of the AA10 family (CAZy classification) were formerlyclassified in the CBM33 family (or “carbohydrate binding module family33”) of the CAZy classification.

The LPMO family AA10 comprises, at the current time, more than about athousand enzymes, identified particularly in bacteria, but also in someeukaryotes and also in some viruses.

The LPMOs of the AA10 family have a structure similar to that of theenzymes of the AA9 family and in particular at least one conservedtyrosine residue in the N-terminal position, which is involved in thebinding with the copper ion. However, in most LPMOs of the AA10 family,one of the other tyrosine residues involved in the axial binding of thecopper ion is replaced with a phenylalanine residue. For these enzymes,an oxidative activity has been demonstrated on chitin and on cellulose.

Preferably, the enzymes of the AA10 family are multimodular and comprisea CBM domain in the N-terminal position. These domains are typicallyCBM2, CBM5, CBM10 and CBM12 domains and also fibronectin type IIImodules.

The AA11 family is characterized by enzymes which carry out an oxidativecleavage in position C₁ on chitin. The enzyme of Aspergillus oryzae willpreferably be chosen (see also Hemsworth et al., Nature Chemical Biology2014(10):122-126—Discovery and characterization of a new family of lyticpolysaccharide monooxygenases).

The AA13 family is characterized by enzymes which carry out an oxidativecleavage in position C₁ on starch. The enzyme of Aspergillus nidulanswill preferably be chosen (Lo Leggio et al., Nat Commun. 2015(22) 6:5961—Structure and boosting activity of a starch-degrading lyticpolysaccharide monooxygenase).

Generally, the step of enzymatic treatment is carried out by means of atleast one LPMO enzyme listed in table 2 hereinafter.

In certain embodiments of the process of the invention, said at leastone enzyme of the LPMO family (advantageously of the AA9 family and/orof the AA10 family) is used in combination with at least one cellulase.

The cellulase is advantageously chosen from at least one endoglucanase(for example one endoglucanase) and/or at least one carbohydratedehydrogenase (advantageously one cellobiose dehydrogenase (CDH)). Thecarbohydrate dehydrogenases can act as an electron donor for the LPMOs.

In practice, said at least one LPMO enzyme used is advantageouslypurified from a culture supernatant of a fungus and/or produced in aheterologous system, in particular in a bacterium, a fungus or a yeast,for example in the Pichia pastoris yeast.

Said at least one LPMO enzyme is mixed with the cellulose-basedsubstrate, so as to allow said at least one enzyme to be brought intocontact with the cellulose fibers.

The step of enzymatic treatment is preferably carried out with gentlestirring, so as to ensure good dispersion of the enzymes within thefibers. This step of enzymatic treatment is for example carried out fora period ranging from 24 h to 72 h (preferably for 48 h).

Preferably, the step of enzymatic treatment is carried out at atemperature ranging from 30 to 45° C.

According to the invention, said at least one LPMO enzyme can be addedto the cellulose-based substrate according to an enzyme/cellulose ratioranging from 1:1000 to 1:50, in particular from 1:500 to 1:50 or from1:100 to 1:50 or else from 1:1000 to 1:500, from 1:500 to 1:100.

Preferably, said at least one LPMO enzyme is used at a concentrationranging from 0.001 to 10 g/l, in particular from 0.1 to 5 g/l, and morepreferably from 0.5 to 5 g/l.

According to one particular embodiment, the cellulose-based substrate issubjected to at least two (or even only to two) successive steps ofenzymatic treatment (in series, advantageously separated by a rinsingstep).

The LPMO(s) used during each of these steps of enzymatic treatment is(are) identical or different; the conditions (in particular theenzyme/substrate ratio) are identical or different between thesesuccessive steps.

In this case, the examples demonstrate that the fibers are entirelydestructured, including at low enzyme/cellulose ratios.

Step(s) of Mechanical Treatment(s)

The pretreated cellulose-based substrate is then subjected to at leastone step of mechanical treatment which is intended to delaminate thecellulose fibers in order to obtain nanocelluloses.

The delamination (also referred to as “fibrillation” or“defibrillation”) consists in separating the cellulose fibers intonanocelluloses, via a mechanical phenomenon.

As demonstrated through the examples below, the oxidative cleavage ofthe cellulose fibers, catalyzed by said at least one LPMO, facilitatesthe delamination of these cellulose fibers during the step of mechanicaltreatment.

This step of mechanical delamination of the cellulose fibers can then becarried out under conditions that are less drastic and therefore lesscostly in terms of energy. Moreover, the use of LPMOs according to theinvention makes it possible to introduce into the cellulose fiberscharged groups which create electrostatic repulsions, withoutcontamination with treatment reagents, such as when TEMPO reagents areused.

The mechanical treatments intended to delaminate cellulose fibers areknown to those skilled in the art and can be implemented in the processof the invention.

Generally, mention may be made of weak mechanical treatments with ahomogenizer-disperser (for example of the Ultra-Turrax type) and/orultrasonic treatments.

Reference may also for example be made to the document of Lavoine N etal. (Carbohydrate Polymers, 2012, (92): 735-64) which describes inparticular (pages 740 to 744) mechanical treatments for preparingmicrofibrilated cellulose (for example cellulose nanofibrils).

Typically, a mechanical treatment can be chosen from mechanicalhomogenization, microfluidization, abrasion or cryomilling treatments.

The homogenization treatment involves passing the pretreatedcellulose-based substrate, typically a cellulose pulp or a liquidsuspension of cellulose, through a narrow space under high pressure (asdescribed for example in patent U.S. Pat. No. 4,486,743).

This homogenization treatment is preferably carried out by means of ahomogenizer of Gaulin type. In such a device, the pretreatedcellulose-based substrate, typically in the form of a cellulosesuspension, is pumped at high pressure and distributed through anautomatic valve with a small orifice. A rapid succession of openings andclosings of the valve subjects the fibers to a considerable drop inpressure (generally of at least 20 MPa) and to a high-speed shear actionfollowed by a high-speed deceleration impact. The passing of thesubstrate through the orifice is repeated (generally from 8 to 10 times)until the cellulose suspension becomes stable. In order to maintain aproduct temperature in a range of from 70 to 80° C. during thehomogenization treatment, cooling water is generally used.

This homogenization treatment can also be carried out by means of adevice of the microfluidizer type (see for example Sisqueira et al.Polymer 2010 2(4): 728-65). In such a device, the cellulose suspensionpasses through a typically “z”-shaped thin chamber (the dimensions ofthe channel of which are generally between 200 and 400 μm) under highpressure (approximately 2070 bar). The high shear rate which is applied(generally greater than 10⁷.s⁻¹) makes it possible to obtain very finenanofibrils. A variable number of passes (for example from 2 to 30, inparticular from 10 to 30 or from 5 to 25, and in particular from 5 to20) with chambers of different sizes can be used, in order to increasethe degree of fibrillation.

The abrasion or milling treatment (see for example Iwamoto S et al.,2007 Applied Physics A89(2): 461-66) is based on the use of a millingdevice capable of exerting shear forces provided by milling stones.

The pretreated cellulose-based substrate, generally in the form of acellulose pulp, is passed between a static milling stone and a rotatingmilling stone, typically at a speed of about 1500 revolutions per minute(rpm). Several passes (generally between 2 and 5) may be required inorder to obtain fibrils of nanometric size.

A device of mixer type (for example as described in Unetani K et al.,Biomacromolecules 2011, 12(2), pp. 348-53) can also be used to producemicrofibrils from pretreated cellulose-based substrate, for example froma suspension of wood fibers.

The cryomilling (or cryocrushing) treatment (Dufresne et al., 1997,Journal of Applied Polymer Science, 64(6): 1185-94) consists in millinga suspension of pretreated cellulose-based substrate frozen beforehandwith liquid nitrogen. The ice crystals formed inside the cells cause thecell membranes to explode and release wall fragments. These processesare generally used for the production of cellulose microfibrils fromagricultural products or residues.

Step(s) of Post-Treatment of the Cellulose-Based Substrate

In certain embodiments, the production process comprises at least onestep of post-treatment of the cellulose-based substrate, carried outafter said substrate has been subjected to the mechanical treatment.

Generally, said at least one post-treatment step aims to increase thedegree of fibrillation of the nanocelluloses obtained and/or to confernew mechanical properties on said nanocelluloses, as a function of theapplications envisioned.

Said at least one post-treatment step can in particular be chosen froman acid treatment, an enzymatic treatment, an oxidation, an acetylation,a silylation, or else a derivatization of certain chemical groups borneby the microfibrils. Reference may also be made, for example, to thedocument by Lavoine N et al (Carbohydrate Polymers, 2012, (92): 735-64)which describes in particular (point 2.3, pages 747 to 748)post-treatments that can be combined with various pretreatments andmechanical treatments of the cellulose fibers.

Nanocelluloses According to the Invention

The process according to the invention thus makes it possible to obtainnanocelluloses, in particular cellulose nanocrystals and/or cellulosenanofibrils.

Contrary to the NFCs obtained after oxidation by chemical reagents ofTEMPO type, the nanocelluloses obtained by means of the process of theinvention are devoid of oxidation reagent residues (namely, for example,sodium bromide, sodium hypochlorite, sodium chlorite, the(2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical (TEMPO), derivatives oranalogs).

Preferably, at the end of the process according to the invention, thenanocelluloses comprise at least one glucose ring (typically severalglucose rings) of which at least one of the carbon atoms in positions C₁and/or C₄, or even also C₆, is oxidized by an oxidative cleavagephenomenon.

The nanocelluloses according to the invention thus comprise glucoserings which are:

-   -   monooxidized on the carbon atom in position C₁, and/or    -   monooxidized on the carbon atom in position C₄, and/or    -   doubly oxidized on the atoms in positions C₁ and C₄.

These glucose rings, oxidized on the atoms in positions C₁ and/or C₄,can also comprise an oxidized carbon atom in position C₆.

The term “oxidized carbon atom” is intended to mean in particular acarbon atom which comprises a carbonyl function, and advantageously alsoa carboxyl function.

The nanocelluloses according to the invention are thus advantageouslynegatively charged, because of the presence of various surface functionsincluding carboxylate functions on the carbons in positions C₁ and/or C₄(contrary to the TEMPO process which results in a specific oxidation ofthe carbon in position C₆).

Experimental Results

1. Tests for cleavage of the cellulose by an LPMO enzyme can be carriedout according to the following protocol:

The cleavage test is carried out at a volume of 300 μl of liquidcontaining 4.4 μM of LPMO enzyme and 1 mM of ascorbate and 0.1%(weight/volume) of powder of phosphoric acid-swollen cellulose(PASO—prepared as described in Wood T M, Methods Enzym 1988, 160: 19-25)in 50 mM of a sodium acetate buffer at pH 4.8 or 5 μM ofcellooligosaccharides (Megazyme, Wicklow, Ireland) in 10 mM of sodiumacetate buffer at pH 4.8.

The enzymatic reaction is carried out in a 2 ml tube incubated in athermomixer (Eppendorf, Montesson, France) at 50° C. and 580 rpm(revolutions per minute).

After incubation for 16 h, the sample is brought to 100° C. for 10minutes in order to stop the enzymatic reaction, then centrifuged at 16000 revolutions per minute (rpm) for 15 minutes at 4° C. in order toseparate the solution fraction from remainder insoluble fraction.

The cleaved products obtained can be analyzed by ion exchangechromatography and/or by mass spectrometry (MALDI-TOF).

2. Preliminary tests were carried out in order to demonstrate theefficiency of the process for producing nanocellulose according to theinvention.

These tests were carried out on a papermaking fiber (cellulose kraftfibers) by means of LPMO enzyme of the AA9 family derived from Podosporaanserina (PaLPMO9E (Genbank CAP67740) and/or PaLPMO9H (GenbankCAP61476)) and produced in a heterologous system in yeast (Pichiapastoris).

The fibers are brought into contact with the enzymes (at a concentrationof 1 g/l and according to enzyme/cellulose ratios of 1:50, 1:100, 1:500and 1:1000) and with ascorbate (2 mM) and then subjected to gentlestirring for 48 hours at 40° C.

The treated fibers are then subjected to a mechanical action with ahomogenizer-disperser (Ultra-Turrax power 500 W, maximum speed for 3minutes), followed by an ultrasonic treatment for 3 minutes.

Compared with the substrates not treated with the enzyme, it is observedthat the defibrillation is facilitated for all of the enzyme/celluloseratios used (FIG. 1B-D—the photos show, qualitatively, thedefibrillation).

In the absence of enzymes, the fibers remain intact and nodefibrillation is noted (see the non-treated control fibers, FIG. 1A).

The dispersions were then analyzed by TEM (Transmission ElectronMicroscopy) and AFM (Atomic Force Microscopy).

In the absence of LPMO enzymes (FIG. 2A), it is noted that very fewstructures are visible on the nanometric scale.

On the other hand, for the fibers treated with the LPMO enzyme,structures of nanometric sizes are easily pinpointed, both in thesupernatant and in the pellets of the experiment. The fibers areentirely destructured, allowing crystalline zones of the fiber to appear(FIG. 2C-D).

The treatment of the fibers with the PaLPMO9E enzyme combined with thesubsequent mechanical treatment (FIGS. 3B and D) produces adefibrillation of the cellulose that is similar to that obtained with anidentical process involving the PaLPMO9H enzyme (see FIG. 3A and C).

Generally, FIGS. 2C, 2D, 3C and 3D unquestionably demonstrate thatnanocelluloses are obtained.

If the fibers that have undergone a first treatment with the LPMO enzymeare again subjected to a second successive treatment with an LPMO enzymeunder the conditions described above, followed by the mechanicaltreatment, the fibers are entirely destructured, including at the lowenzyme/cellulose ratios (that is to say the 1/500 and 1/1000 ratios)(FIG. 4).

The AFM photos for the PaLPMO9H enzyme were analyzed using the WSxMsoftware in order to characterize the height profile (FIG. 5A) and thesize distribution (FIG. 5B) of the nanocelluloses.

This analysis shows that, at the 1:50 enzyme/cellulose ratio, thenanocelluloses have a diameter of less than 100 nm.

This result confirms that the products obtained by means of the processaccording to the invention are nanocelluloses.

TABLE 1 Listed fungal enzymes of the AA9 family (CAZy classification)Name Organism GenBank ref. Uniprot ref. Cel1 Agaricus bisporusAAA53434.1 Q00023 D649 AfA5C5.025 Aspergillus CAF31975.1 Q6MYM8fumigatus endoglucanase/CMCase Aspergillus AFJ54163.1 (Eng61) fumigatusMKU1 endo-β-1,4-glucanase B (EgIB; Aspergillus BAB62318.1 Q96WQ9AkCel61A) (Cel61A) kawachii NBRC4308 AN1041.2 Aspergillus EAA65609.1C8VTW9 nidulans FGSC A4 Q5BEI9 AN3511.2 Aspergillus EAA59072.1 Q5B7G9nidulans FGSC A4 AN9524.2 Aspergillus EAA66740.1 C8VI93 nidulans FGSC A4CBF83171.1 Q5AQA6 AN7891.2 Aspergillus EAA59545.1 Q5AUY9 nidulans FGSCA4 AN6428.2 Aspergillus EAA58450.1 C8V0F9 nidulans FGSC A4 Q5AZ52AN3046.2 Aspergillus EAA63617.1 C8VIS7 nidulans FGSC A4 Q5B8T4 AN3860.2(EgIF) Aspergillus EAA59125.1 C8V6H2 nidulans FGSC A4 Q5B6H0endo-β-1,4-glucanase Aspergillus EAA64722.1 Q5BCX8 (AN1602.2) nidulansFGSC A4 ABF50850.1 AN2388.2 Aspergillus EAA64499.1 C8VNP4 nidulans FGSCA4 Q5BAP2 An04g08550 Aspergillus niger CAK38942.1 A2QJX0 CBS 513.88An08g05230 Aspergillus niger CAK45495.1 A2QR94 CBS 513.88 An12g02540Aspergillus niger CAK41095.1 A2QYU6 CBS 513.88 An12g04610 Aspergillusniger CAK97151.1 A2QZE1 CBS 513.88 An14g02670 Aspergillus nigerCAK46515.1 A2R313 CBS 513.88 An15g04570 Aspergillus niger CAK97324.1A2R5J9 CBS 513.88 An15g04900 Aspergillus niger CAK42466.1 A2R5N0 CBS513.88 AO090005000531 Aspergillus oryzae BAE55582.1 Q2US83 RIB40AO090001000221 Aspergillus oryzae BAE56764.1 Q2UNV1 RIB40 AO090023000056Aspergillus oryzae BAE58643.1 Q2UIH2 RIB40 AO090023000159 Aspergillusoryzae BAE58735.1 Q2UI80 RIB40 AO090023000787 Aspergillus oryzaeBAE59290.1 Q2UGM5 RIB40 AO090012000090 Aspergillus oryzae BAE60320.1Q2UDP5 RIB40 AO090138000004 Aspergillus oryzae BAE64395.1 Q2U220 RIB40AO090103000087 Aspergillus oryzae BAE65561.1 Q2TYW2 RIB40 Cel6 (E6)Bipolaris maydis AAM76663.1 Q8J0H7 C4 glycoside hydrolase family 61Botryotinia CCD34368.1 protein (Bofut4_p103280.1) fuckeliana T4glycoside hydrolase family 61 Botryotinia CCD47228.1 protein(Bofut4_p003870.1) fuckeliana T4 glycoside hydrolase family 61Botryotinia CCD48549.1 protein (Bofut4_p109330.1) fuckeliana T4glycoside hydrolase family 61 Botryotinia CCD50139.1 protein(Bofut4_p025380.1) fuckeliana T4 glycoside hydrolase family 61Botryotinia CCD50144.1 protein (Bofut4_p025430.1) fuckeliana T4glycoside hydrolase family 61 Botryotinia CCD51504.1 protein(Bofut4_p018100.1) fuckeliana T4 glycoside hydrolase family 61Botryotinia CCD49290.1 protein (Bofut4_p031660.1) fuckeliana T4glycoside hydrolase family 61 Botryotinia CCD52645.1 protein(Bofut4_p000920.1) fuckeliana T4 BofuT4P143000045001 BotryotiniaCCD50451.2 fuckeliana T4 CCD50451.1 ORF Chaetomium AGY80102.1thermophilum CT2 ORF (fragment) Chaetomium AGY80103.1 thermophilum CT2ORF (fragment) Chaetomium AGY80104.1 thermophilum CT2 ORF (fragment)Chaetomium AGY80105.1 thermophilum CT2 cellobiohydrolase family proteinChaetomium AGY80103.1 61, partial (Cbh61-2) (fragment) thermophilum CT2cellobiohydrolase family protein Chaetomium AGY80104.1 61, partial(Cbh61-3) (fragment) thermophilum CT2 cellobiohydrolase family proteinChaetomium AGY80105.1 61, partial (Cbh61-4) (fragment) thermophilum CT2ORF (possible fragment) Colletotrichum CAQ16278.1 B5WYD8 graminicola M2ORF Colletotrichum CAQ16206.1 B5WY66 graminicola M2 ORF ColletotrichumCAQ16208.1 B5WY68 graminicola M2 ORF Colletotrichum CAQ16217.1 B5WY77graminicola M2 unnamed protein product Coprinopsis CAG27578.1 cinereaCGB_A6300C Cryptococcus ADV19810.1 bacillisporus WM276 CNAG_00601Cryptococcus AFR92731.1 neoformans var. AFR92731.2 grubii H99(Cryne_H99_1) Cel1 Cryptococcus AAC39449.1 O59899 neoformans var.neoformans CNA05840 (Cel1) Cryptococcus AAW41121.1 F5HH24 neoformansvar. neoformans JEC21 (Cryne_JEC21_1) ORF (fragment) FlammulinaADX07320.1 velutipes KACC 42777 FFUJ_12340 Fusarium fujikuroi CCT72465.1IMI 58289 (Fusfu1) FFUJ_13305 Fusarium fujikuroi CCT67119.1 IMI 58289(Fusfu1) FFUJ_07829 Fusarium fujikuroi CCT69268.1 IMI 58289 (Fusfu1)FFUJ_12621 Fusarium fujikuroi CCT72729.1 IMI 58289 (Fusfu1) FFUJ_12840Fusarium fujikuroi CCT72942.1 IMI 58289 (Fusfu1) FFUJ_09373 Fusariumfujikuroi CCT73805.1 IMI 58289 (Fusfu1) FFUJ_10599 Fusarium fujikuroiCCT74544.1 IMI 58289 (Fusfu1) FFUJ_10643 Fusarium fujikuroi CCT74587.1IMI 58289 (Fusfu1) FFUJ_14514 Fusarium fujikuroi CCT67584.1 IMI 58289(Fusfu1) FFUJ_11399 Fusarium fujikuroi CCT75380.1 IMI 58289 (Fusfu1)FFUJ_14514 Fusarium fujikuroi CCT67584.1 IMI 58289 FFUJ_11399 Fusariumfujikuroi CCT75380.1 IMI 58289 FFUJ_04652 Fusarium fujikuroi CCT64153.1IMI 58289 (Fusfu1) FFUJ_03777 Fusarium fujikuroi CCT64954.1 IMI 58289(Fusfu1) FFUJ_04940 Fusarium fujikuroi CCT63889.1 IMI 58289 (Fusfu1)Sequence 122805 from patent Fusarium ABT35335.1 U.S. Pat. No. 7,214,786graminearum FG03695.1 (Cel61E) Fusarium XP_383871.1 graminearum PH-1unnamed protein product Fusarium CEF78545.1 graminearum PH-1 unnamedprotein product Fusarium CEF74901.1 graminearum PH-1 unnamed proteinproduct Fusarium CEF78472.1 graminearum PH-1 unnamed protein productFusarium CEF86346.1 graminearum PH-1 unnamed protein product FusariumCEF87450.1 graminearum PH-1 unnamed protein product Fusarium CEF85876.1graminearum PH-1 unnamed protein product Fusarium CEF86254.1 graminearumPH-1 unnamed protein product Fusarium CEF87657.1 graminearum PH-1unnamed protein product Fusarium CEF76256.1 graminearum PH-1 unnamedprotein product Fusarium CEF78876.1 graminearum PH-1 unnamed proteinproduct Fusarium CEF79735.1 graminearum PH-1 unnamed protein productFusarium CEF74460.1 graminearum PH-1 unnamed protein product FusariumCEF84640.1 graminearum PH-1 endo-β-1,4-glucanase (Cel61G) GloeophyllumAEJ35168.1 trabeum GH61D Heterobasidion AFO72234.1 parviporum GH61BHeterobasidion AFO72233.1 parviporum GH61A Heterobasidion AFO72232.1parviporum GH61F Heterobasidion AFO72235.1 parviporum GH61GHeterobasidion AFO72236.1 parviporum GH61H Heterobasidion AFO72237.1parviporum GH61I Heterobasidion AFO72238.1 parviporum GH61JHeterobasidion AFO72239.1 parviporum unnamed protein product Humicolainsolens CAG27577.1 endoglucanase IV (EgiV) Hypocrea orientalisAFD50197.1 EU7-22 GH61A (GH61A) Lasiodiplodia CAJ81215.1 theobromae CBS247.96 GH61B (GH61B) Lasiodiplodia CAJ81216.1 theobromae CBS 247.96GH61C (GH61C) Lasiodiplodia CAJ81217.1 theobromae CBS 247.96 GH61D(GH61D) Lasiodiplodia CAJ81218.1 theobromae CBS 247.96 ORF LeptosphaeriaCBX91313.1 E4ZJM8 maculans v23.1.3 ORF Leptosphaeria CBX93546.1 E4ZQ11maculans v23.1.3 ORF Leptosphaeria CBX94224.1 E4ZS44 maculans v23.1.3ORF Leptosphaeria CBX94532.1 E4ZSU4 maculans v23.1.3 ORF LeptosphaeriaCBX94572.1 E4ZSY4 maculans v23.1.3 ORF Leptosphaeria CBX95655.1 E4ZVM9maculans v23.1.3 ORF Leptosphaeria CBX96476.1 E4ZZ41 maculans v23.1.3ORF Leptosphaeria CBX96550.1 E4ZYM4 maculans v23.1.3 ORF LeptosphaeriaCBX96949.1 E5A089 maculans v23.1.3 ORF Leptosphaeria CBX97718.1 E5A201maculans v23.1.3 ORF Leptosphaeria CBX98126.1 E5A3B3 maculans v23.1.3ORF Leptosphaeria CBY01974.1 E5AFI5 maculans v23.1.3 ORF LeptosphaeriaCBY02242.1 E5ACP0 maculans v23.1.3 ORF Leptosphaeria CBX91667.1 E4ZK72maculans v23.1.3 ORF Leptosphaeria CBX93965.1 E4ZQA3 maculans v23.1.3ORF Leptosphaeria CBX98254.1 E5A3P1 maculans v23.1.3 ORF (fragment)Leptosphaeria CBY00196.1 E5A955 maculans v23.1.3 ORF LeptosphaeriaCBY01204.1 E5AC13 maculans v23.1.3 predicted protein LeptosphaeriaCBY01256.1 E5ADG7 (Lema_p000430.1) (fragment) maculans v23.1.3 ORF(fragment) Leptosphaeria CBY01257.1 E5ADG8 maculans v23.1.3 lyticpolysaccharide Leucoagaricus CDJ79823.1 monooxygenase gongylophorusAe322 MG05364.4 Magnaporthe EAA54572.1 grisea 70-15 XP_359989.1 (Maggr1)MG07686.4 Magnaporthe EAA53409.1 G4N3E5 grisea 70-15 XP_367775.1(Maggr1) MG07300.4 Magnaporthe EAA56945.1 G4MUY8 grisea 70-15XP_367375.1 (Maggr1) MG08020.4 Magnaporthe EAA57051.1 grisea 70-15XP_362437.1 (Maggr1) MG08254.4 Magnaporthe EAA57285.1 G4MXC7 grisea70-15 XP_362794.1 (Maggr1) MG08066.4 (fragment) Magnaporthe EAA57097.1G4MXS5 grisea 70-15 XP_362483.1 (Maggr1) MG04547.4 MagnaportheEAA50788.1 G4MS66 grisea 70-15 XP_362102.1 (Maggr1) MG08409.4Magnaporthe EAA57439.1 G4MVX4 grisea 70-15 XP_362640.1 (Maggr1)MG09709.4 Magnaporthe EAA49718.1 G4NAI5 grisea 70-15 XP_364864.1(Maggr1) MG06069.4 Magnaporthe EAA52941.1 G4N560 grisea 70-15XP_369395.1 (Maggr1) MG09439.4 Magnaporthe EAA51422.1 G4NHT8 grisea70-15 XP_364487.1 (Maggr1) MG06229.4 Magnaporthe EAA56258.1 grisea 70-15XP_369714.1 (Maggr1) MG07631.4 Magnaporthe EAA53354.1 G4N2Z0 grisea70-15 XP_367720.1 (Maggr1) MGG_06621 Magnaporthe XP_003716906.1 grisea70-15 XP_370106.1 (Maggr1) MGG_12696 Magnaporthe XP_003721313.1 grisea70-15 (Maggr1) MGG_02502 Magnaporthe XP_003709306.1 grisea 70-15EAA54517.1 (Maggr1) XP_365800.1 MGG_04057 Magnaporthe XP_003719782.1grisea 70-15 EAA50298.1 (Maggr1) XP_361583.1 MGG_13241 MagnaportheXP_003711808.1 grisea 70-15 (Maggr1) MGG_13622 MagnaportheXP_003717521.1 grisea 70-15 (Maggr1) MGG_07575 MagnaportheXP_003711490.1 grisea 70-15 EAA53298.1 (Maggr1) XP_367664.1 MGG_11948Magnaporthe XP_003709110.1 grisea 70-15 (Maggr1) MGG_16080 (fragment)Magnaporthe XP_003709033.1 grisea 70-15 (Maggr1) MGG_16043 (fragment)Magnaporthe XP_003708922.1 grisea 70-15 (Maggr1) MGG_12733 (probablefragment) Magnaporthe XP_003716689.1 grisea 70-15 (Maggr1)copper-dependent Malbranchea CCP37674.1 polysaccharide monooxygenasescinnamomea CBS (Gh61) (fragment) 115.68 copper-dependent MelanocarpusCCP37668.1 polysaccharide monooxygenases albomyces CBS (Gh61) (fragment)638.94 copper-dependent Myceliophthora CCP37667.1 polysaccharidemonooxygenases fergusii CBS (Gh61) (fragment) 406.69 MYCTH_2112799Myceliophthora AEO61257.1 thermophila ATCC 42464 MYCTH_79765Myceliophthora AEO56016.1 thermophila ATCC 42464 MYCTH_110651Myceliophthora AEO54509.1 thermophila ATCC 42464 MYCTH_2298502Myceliophthora AEO55082.1 thermophila ATCC 42464 MYCTH_2299721Myceliophthora AEO55652.1 thermophila ATCC 42464 MYCTH_2054500Myceliophthora AEO55776.1 thermophila ATCC 42464 MYCTH_111088Myceliophthora AEO56416.1 thermophila ATCC 42464 β-glycan-cleavingenzyme Myceliophthora AEO56542.1 (StCel61a; MYCTH_46583) thermophilaATCC (Cel61A) 42464 MYCTH_2301632 Myceliophthora AEO56547.1 thermophilaATCC 42464 MYCTH_100518 Myceliophthora AEO56642.1 thermophila ATCC 42464lytic polysaccharide Myceliophthora AEO56665.1 monooxygenases (active onthermophila ATCC cellulose) (MYCTH_92668) 42464 MYCTH_2060403Myceliophthora AEO58412.1 thermophila ATCC 42464 MYCTH_2306673Myceliophthora AEO58921.1 thermophila ATCC 42464 MYCTH_116175 (fragment)Myceliophthora AEO59482.1 thermophila ATCC 42464 MYCTH_96032Myceliophthora AEO59823.1 thermophila ATCC 42464 MYCTH_103537Myceliophthora AEO59836.1 thermophila ATCC 42464 MYCTH_55803Myceliophthora AEO59955.1 thermophila ATCC 42464 lytic polysaccharideMyceliophthora AEO60271.1 monooxygenases (active on thermophila ATCCcellulose) (MYCTH_112089) 42464 MYCTH_85556 Myceliophthora AEO61304.1thermophila ATCC 42464 MYCTH_2311323 Myceliophthora AEO61305.1thermophila ATCC 42464 MYCTH_47093 (fragment) Myceliophthora AEO56498.1thermophila ATCC 42464 MYCTH_80312 Myceliophthora AEO58169.1 thermophilaATCC 42464 lytic polysaccharide Neurospora crassa CAD21296.1 Q1K8B6monooxygenase (active on OR74A EAA32426.1 Q8WZQ2 cellulose) (PMO-XP_326543.1 2; NcLPMO9D; GH61- 4; NCU01050) (LPMO9D) lyticpolysaccharide Neurospora crassa CAD70347.1 Q1K4Q1 monooxygenase (activeon OR74A EAA26656.1 Q873G1 cellulose) (PMO- XP_322586.1 03328; NcLPMO9F;GH61- 6; NCU03328) (LPMO9F) lytic polysaccharide Neurospora crassaCAE81966.1 Q7SHD9 monooxygenase (PMO-01867; OR74A EAA36262.1 NcLPMO9J;GH61-10; XP_329057.1 NCU01867; B13N4.070) (LPMO9J) NCU02344.1(B23N11.050) Neurospora crassa CAF05857.1 Q7S411 OR74A EAA30230.1XP_331120.1 lytic polysaccharide Neurospora crassa EAA33178.1 Q7SA19monooxygenase (active on OR74A XP_328604.1 cellulose) (PMO- 3; NcLPMO9M;GH61-13; NcPMO- 3; NCU07898) (LPMO9M) NCU05969.1 Neurospora crassaEAA29347.1 Q7S1V2 OR74A XP_325824.1 lytic polysaccharide Neurosporacrassa EAA36362.1 Q7SHI8 monooxygenase (active on OR74A XP_330104.1cellulose and cellooligosaccharides) (PMO- 02916; NcLPMO9C; GH61- 3;NCU02916) (LPMO9C) lytic polysaccharide Neurospora crassa EAA29018.1Q7S111 monooxygenase (active on OR74A XP_328466.1 cellulose) (GH61-2;NCU07760) NCU07520.1 Neurospora crassa EAA29132.1 Q7S1A0 OR74AXP_327806.1 lytic polysaccharide Neurospora crassa EAA30263.1 Q7S439monooxygenase (active on OR74A XP_331016.1 cellulose) (GH61-1; NCU02240)lytic polysaccharide Neurospora crassa EAA34466.1 Q7SCJ5 monooxygenase(active on OR74A XP_325016.1 cellulose) (NCU00836) lytic polysaccharideNeurospora crassa EAA26873.1 Q7RWN7 monooxygenase (active on OR74AXP_330877.1 cellulose) (PMO- 08760; NcLPMO9E; GH61- 5; NCU08760)(LPMO9E) NCU07974.1 Neurospora crassa EAA33408.1 Q7SAR4 OR74AXP_328680.1 NCU03000.1 (B24P7.180) Neurospora crassa EAA36150.1 Q7RV41OR74A CAB97283.2 Q9P3R7 XP_330187.1 cellulose monooxygenase PenicilliumAIO06742.1 oxalicum GZ-2 Pc12g13610 Penicillium CAP80988.1 B6H016chrysogenum Wisconsin 54-1255 (PenchWisc1_1) Pc13g07400 PenicilliumCAP91809.1 B6H3U0 chrysogenum Wisconsin 54-1255 (PenchWisc1_1)Pc13g13110 Penicillium CAP92380.1 B6H3A3 chrysogenum Wisconsin 54-1255(PenchWisc1_1) Pc20g11100 Penicillium CAP86439.1 B6HG02 chrysogenumWisconsin 54-1255 (PenchWisc1_1) Cel61 (Cel61A) Phanerochaete AAM22493.1Q8NJI9 chrysosporium BKM-F-1767 Lytic polysaccharide mono- PhanerochaeteBAL43430.1 oxygenase active on cellulose chrysosporium K-3 (Gh61D;PcGH61D) PIIN_01487 Piriformospora CCA67659.1 indica (Pirin1) PIIN_02110Piriformospora CCA68244.1 indica (Pirin1) PIIN_03975 PiriformosporaCCA70035.1 indica (Pirin1) PIIN_04357 Piriformospora CCA70418.1 indica(Pirin1) PIIN_04637 Piriformospora CCA70703.1 indica (Pirin1) PIIN_06117Piriformospora CCA72182.1 indica (Pirin1) PIIN_06118 PiriformosporaCCA72183.1 indica (Pirin1) PIIN_06127 Piriformospora CCA72192.1 indica(Pirin1) PIIN_06155 Piriformospora CCA72220.1 indica (Pirin1) PIIN_07098Piriformospora CCA73144.1 indica (Pirin1) PIIN_07105 PiriformosporaCCA73151.1 indica (Pirin1) PIIN_08199 Piriformospora CCA74246.1 indica(Pirin1) PIIN_08783 Piriformospora CCA74814.1 indica (Pirin1) PIIN_09022Piriformospora CCA75037.1 indica (Pirin1) PIIN_00566 (fragment)Piriformospora CCA66803.1 indica (Pirin1) PIIN_01484 PiriformosporaCCA67656.1 indica (Pirin1) PIIN_01485 (fragment) PiriformosporaCCA67657.1 indica (Pirin1) PIIN_01486 (fragment) PiriformosporaCCA67658.1 indica (Pirin1) PIIN_04356 Piriformospora CCA70417.1 indica(Pirin1) PIIN_05699 (fragment) Piriformospora CCA71764.1 indica (Pirin1)PIIN_06156 Piriformospora CCA72221.1 indica (Pirin1) PIIN_08402Piriformospora CCA74449.1 indica (Pirin1) PIIN_10315 (fragment)Piriformospora CCA76320.1 indica (Pirin1) PIIN_10660 (fragment)Piriformospora CCA76671.1 indica (Pirin1) PIIN_00523 (fragment)Piriformospora CCA77877.1 indica (Pirin1) Putative Glycoside HydrolasePodospora CDP30131.1 B2AL94 Family 61 anserina S mat+ CAP64732.1(Podan2) Putative Glycoside Hydrolase Podospora CDP30928.1 B2B346 Family61 anserina S mat+ CAP71532.1 (Podan2) Pa_1_500 Podospora CAP59702.1B2A9F5 anserina S mat+ CDP22345.1 (Podan2) Pa_4_350 Podospora CAP61395.1B2AD80 anserina S mat+ CDP27750.1 (Podan2) Pa_4_1020 PodosporaCAP61476.1 B2ADG1 anserina S mat+ CDP27830.1 (Podan2) Pa_0_270 PodosporaCAP61650.1 B2ADY5 anserina S mat+ CDP28001.1 (Podan2) Pa_5_8940Podospora CAP64619.1 B2AKU6 anserina S mat+ CDP30017.1 (Podan2)Pa_5_4100 (fragment) Podospora CAP64865.1 B2ALM7 anserina S mat+CDP29378.1 (Podan2) Pa_5_6950 Podospora CAP65111.1 B2AMI8 anserina Smat+ CDP29800.1 (Podan2) Pa_5_10660 Podospora CAP65855.1 B2APD8 anserinaS mat+ CDP30283.1 (Podan2) Pa_5_10760 Podospora CAP65866.1 B2APE9anserina S mat+ CDP30272.1 (Podan2) Pa_5_11630 (fragment) PodosporaCAP65971.1 B2API9 anserina S mat+ CDP30166.1 (Podan2) Pa_4_7570Podospora CAP66744.1 B2ARG6 anserina S mat+ CDP28479.1 (Podan2)Pa_1_21900 (fragment) Podospora CAP67176.1 B2AS05 anserina S mat+CDP24589.1 (Podan2) Pa_1_22040 Podospora CAP67190.1 B2AS19 anserina Smat+ CDP24603.1 (Podan2) Pa_1_22150 (fragment) Podospora CAP67201.1B2AS30 anserina S mat+ CDP24614.1 (Podan2) Pa_6_11220 PodosporaCAP67466.1 B2ASU3 anserina S mat+ CDP30332.1 (Podan2) Pa_6_11370Podospora CAP67481.1 B2ASV8 anserina S mat+ CDP30347.1 (Podan2)Pa_6_11470 Podospora CAP67493.1 B2ASX0 anserina S mat+ CDP30359.1(Podan2) Pa_1_16300 Podospora CAP67740.1 B2ATL7 anserina S mat+CDP23998.1 (Podan2) Pa_7_5030 Podospora CAP68173.1 B2AUV0 anserina Smat+ CDP31642.1 (Podan2) Pa_7_3770 Podospora CAP68309.1 B2AV86 anserinaS mat+ CDP31780.1 (Podan2) Pa_7_3390 Podospora CAP68352.1 B2AVC8anserina S mat+ CDP31823.1 (Podan2) lytic polysaccharide mono- PodosporaCAP68375.1 B2AVF1 oxygenase active on cellulose anserina S mat+CDP31846.1 (Gh61B; Pa_7_3160)(Gh61B) (Podan2) Pa_6_7780 PodosporaCAP71839.1 B2B403 anserina S mat+ CDP31230.1 (Podan2) Pa_2_1700Podospora CAP72740.1 B2B4L5 anserina S mat+ CDP25137.1 (Podan2)Pa_2_4860 Podospora CAP73072.1 B2B5J7 anserina S mat+ CDP25472.1(Podan2) lytic polysaccharide mono- Podospora CAP73254.1 B2B629oxygenase active on cellulose anserina S mat+ CDP25655.1 (Gh61A;Pa_2_6530)(Gh61A) (Podan2) Pa_2_7040 Podospora CAP73311.1 B2B686anserina S mat+ CDP25714.1 (Podan2) Pa_2_7120 Podospora CAP73320.1B2B695 anserina S mat+ CDP25723.1 (Podan2) Pa_3_190 Podospora CAP61048.1B2AC83 anserina S mat+ CDP26500.1 (Podan2) Pa_3_2580 PodosporaCAP70156.1 B2AZV6 anserina S mat+ CDP26748.1 (Podan2) Pa_3_3310Podospora CAP70248.1 B2AZD4 anserina S mat+ CDP26841.1 (Podan2)endo-β-1,4-glucanase (Egl1; Pyrenochaeta AEV53599.1 PIEGL1) lycopersiciISPaVe ER 1211 copper-dependent Rasamsonia CCP37669.1 polysaccharidemonooxygenases byssochlamydoides (Gh61) (fragment) CBS 151.75copper-dependent Remersonia CCP37675.1 polysaccharide monooxygenasesthermophila CBS (Gh61) (fragment) 540.69 RHTO0S_28e01816g RhodosporidiumCDR49619.1 toruloides CECT1137 copper-dependent Scytalidium CCP37676.1polysaccharide monooxygenases indonesiacum CBS (Gh61) (fragment) 259.81SMU2916 (fragment) Sordaria CAQ58424.1 C1KU36 macrospora k-hell lyticpolysaccharide mono- Thermoascus ABW56451.1 oxygenase active oncellulose aurantiacus ACS05720.1 copper-dependent Thermoascus CCP37673.1polysaccharide monooxygenases aurantiacus CBS (Gh61) (fragment) 891.70ORF Thermoascus AGO68294.1 aurantiacus var. levisporus copper-dependentThermomyces CCP37672.1 polysaccharide monooxygenases dupontii CBS (Gh61)(fragment) 236.58 copper-dependent Thermomyces CCP37678.1 polysaccharidemonooxygenases lanuginosus CBS (Gh61) (fragment) 632.91 unnamed proteinproduct Thielavia terrestris CAG27576.1 THITE_2106556 Thielaviaterrestris AEO62422.1 NRRL 8126 THITE_2116536 Thielavia terrestrisAEO67662.1 NRRL 8126 THITE_2040127 Thielavia terrestris AEO64605.1 NRRL8126 THITE_2119040 Thielavia terrestris AEO69044.1 NRRL 8126THITE_115795 Thielavia terrestris AEO64177.1 NRRL 8126 THITE_2110890Thielavia terrestris AEO64593.1 NRRL 8126 THITE_2112626 Thielaviaterrestris AEO65532.1 NRRL 8126 THITE_2076863 Thielavia terrestrisAEO65580.1 NRRL 8126 THITE_170174 Thielavia terrestris AEO66274.1 NRRL8126 THITE_2044372 Thielavia terrestris AEO67396.1 NRRL 8126THITE_2170662 Thielavia terrestris AEO68023.1 NRRL 8126 THITE_128130Thielavia terrestris AEO68157.1 NRRL 8126 THITE_2145386 Thielaviaterrestris AEO68577.1 NRRL 8126 THITE_2054543 Thielavia terrestrisAEO68763.1 NRRL 8126 THITE_2059487 Thielavia terrestris AEO71031.1 NRRL8126 THITE_2142696 Thielavia terrestris AEO67395.1 NRRL 8126 THITE_43665Thielavia terrestris AEO69043.1 NRRL 8126 THITE_2085430 (fragment)Thielavia terrestris AEO63926.1 NRRL 8126 THITE_2122979 Thielaviaterrestris XP_003657366.1 NRRL 8126 cellulase-enhancing Thielaviaterrestris ACE10231.1 factor (GH61B) NRRL 8126 Sequence 4 from patentU.S. Pat. No. Thielavia terrestris ACE10232.1 7,361,495 (GH61C) NRRL8126 Sequence 4 from patent U.S. Pat. No. Thielavia terrestrisACE10232.1 7,361,495 (GH61C) NRRL 8126 Sequence 6 from patent U.S. Pat.No. Thielavia terrestris ACE10233.1 7,361,495 (GH61D) NRRL 8126 Sequence6 from patent U.S. Pat. No. Thielavia terrestris ACE10233.1 7,361,495(GH61D) NRRL 8126 lytic polysaccharide mono- Thielavia terrestrisAEO71030.1 oxygenase active on cellulose NRRL 8126 ACE10234.1 (131562;TtGH61E)(GH61E) Sequence 10 from patent U.S. Pat. No. Thielaviaterrestris ACE10235.1 7,361,495 (GH61G) NRRL 8126 Sequence 10 frompatent U.S. Pat. No. Thielavia terrestris ACE10235.1 7,361,495 (GH61G)NRRL 8126 Lytic polysaccharide mono- Trichoderma reesei AAP57753.1Q7Z9M7 oxygenase active on cellulose QM6A ABH82048.1 (EG7;HjGH61B)(Cel61B = GH61B) ACK19226.1 ACR92640.1 endo-γ-1,4-glucanase IVTrichoderma reesei CAA71999.1 O14405 (EGIV; Egl4; EG4) (Cel61A) RUTC-30endoglucanase Trichoderma ADB89217.1 D3JTC4 (EnGluIV; EndoGluIV)saturnisporum endoglucanase IV (EgIV; EG IV) Trichoderma sp. ACH92573.1B5TYI4 SSL endoglucanase VII (EgvII) Trichoderma viride ACD36971.1B4YEW1 AS 3.3711 endoglucanase IV (EgIV) Trichoderma viride ADJ57703.1B4YEW3 AS 3.3711 ACD36973.1 D9IXC6 AAA12YM05FL uncultured CCA94933.1eukaryote AAA2YG01FL uncultured CCA94930.1 eukaryote AAA15YI10FLuncultured CCA94931.1 eukaryote AAA21YH11FL uncultured CCA94932.1eukaryote ABA3YP05FL uncultured CCA94934.1 eukaryote endoglucanase II(EgII) Volvariella AFP23133.1 volvacea endoglucanase II (EgII)Volvariella AAT64005.1 Q6E5B4 volvacea V14 Unknown Zea mays B73ACF86151.1 unknown (ZM_BFc0036G02) Zea mays B73 ACF78974.1 B4FA31ACR36748.1

TABLE 2 LPMOs (AA9, AA10 and AA11 families of the CAZy classification)Uniprot GenBank Substrate Known Organism ref. ref. Other namesspecificity selectivity Modularity fungi A. oryzae Q2UA85 BAE61530AoAA11 chitin C1 AA11- X278 A. nidulans C8VGF8 EAA62623.1 AnAA13 starchC1 AA13- CBM20 M. thermophila G2QI82 AEO60271 MYCTH_112089 cellulose C1AA9 M. thermophila G2QAB5 AEO56665 MYCTH_92668 cellulose C1 AA9 N.crassa Q7RWN7 EAA26873 NcLPMO9E cellulose C1 AA9- CBM1 N. crassa Q1K8B6EAA32426 NcLPMO9D cellulose C4 AA9 Q8WZQ2 CAD21296 N. crassa Q7SA19EAA33178 NcLPMO9M cellulose C1, C4 AA9 N. crassa Q7SHI8 EAA36362NcLPMO9C cellulose C4 AA9- hemi- CBM1 cellulose N. crassa Q1K4Q1EAA26656 NcLPMO9F cellulose C1 AA9 CAD70347 N. crassa Q7SCJ5 EAA34466NcU00836 cellulose C1 AA9- CBM1 N. crassa Q7SCE9 EAA34371.2 NcAA13starch C1 AA13- CBM20 N. crassa Q7S439 EAA30263 NcU02240 cellulose C4AA9- CBM1 N. crassa Q7S111 EAA29018 NcU07760 cellulose C1, C4 AA9- CBM1P. chrysosporium H1AE14 BAL43430 PcLPMO9D cellulose C1 AA9 P. anserinaB2B629 CAP73254 PaGH61A cellulose C1^(a), C4^(a) AA9- PaLMPOB CBM1 P.anserina B2AVF1 CAP68375 PaGH61B cellulose C1, C4 AA9- PaLMPO9A CBM1 P.anserina B2ARG6 CAP66744 PaLPMO9D cellulose C1 AA9 CBM1 P. anserinaB2ATL7 CAP67740 PaLPMO9E cellulose C1 AA9 CBM1 P. anserina B2B403CAP71839 PaLPMO9F cellulose n.d AA9 CBM1 P. anserina B2B5J7 CAP73072PaLPMO9G cellulose n.d AA9 CBM1 P. anserina B2ADG1 CAP64476 PaLPMO9Hcellulose C1, C4 AA9 CBM1 T. aurantiacus G3XAP7 ABW56451 TaGH61Acellulose C1 AA9 T. terrestris G2RGE5 AEO71030 cellulose n.d. AA9 T.reesei Q7Z9M7 AAP57753 cellulose n.d. AA9 T. reesei O14405 CAA71999Cel61A cellulose n.d. AA9 Bacteria Bacillus E1UUV3 CBI42985 n.d. n.d.AA10 amyloliquefaciens Burkholderia Q3JY22 ABA49030 BURPS1710b_0114 n.d.n.d. AA10 pseudomallei (BpAA10A) 1710b Bacillus Q62YN7 AAU22121 chitinC1 AA10 licheniformes Caldibacillus Q9RFX5 AAF22274 β-1,4- n.d. n.d.AA10 cellulovorans mannanase (ManA) Enterococcus Q838S1 AAO80225EfLPMO10A chitin C1 AA10 faecalis Hahella Q2SNS3 ABC27701 LPMO cellulosend AA10 chejuensis (HcAA10- 2; HCH_00807) Serratia O83009 AAU88202SmLPMO10A chitin C1 AA10 marcescens Streptomyces Q9RJC1 CAB61160ScLPMO10B cellulose C1, C4 AA10 coelicolor chitin Streptomyces Q9RJY2CAB61600 ScLPMO10C cellulose C1 AA10- coelicolor CBM2 ThermobifidaQ47QG3 AAZ55306 TfLPMO10A cellulose C1, C4 AA10 fusca chitinThermobifida Q47PB9 AAZ55700 TfLPMO10B cellulose C1 AA10- fusca CBM2 V.cholerae Q9KLD5 AAF96709 VcLPMO10B n.d. n.d. AA10 O1 The term “substratespecificity” is intended to mean the type of substrate cleaved(oxidative cleavage) by the corresponding LPMO enzyme. The term “knownselectivity” is intended to mean the carbon of the glucose ring oxidizedby the corresponding LPMO enzyme. The term “modularity” is intended tomean the CAZy class (AA9, 10 or 11) of the enzyme and the known presenceof a conserved domain (CBM or X278).

The invention claimed is:
 1. A process for producing nanocelluloses froma cellulose-based substrate comprising cellulose fibers, said processcomprising the following successive steps: one or more step(s) ofenzymatic treatment of said cellulose-based substrate, by bringing itinto contact with at least one cleavage enzyme consisting of enzymesbelonging to the lytic polysaccharide monooxygenase (LPMO) familycapable of carrying out an oxidative cleavage of said cellulose fibersin the presence of a donor electron, wherein said at least one LPMOenzyme is purified from a culture supernatant of a fungus and/orproduced in a heterologous system, wherein said at least one LPMOintroduces, into the cellulose fibers, charged groups which createelectrostatic repulsions, then at least one step of mechanical treatmentof said cellulose-based substrate subjected to said one or more step(s)of enzymatic treatment, wherein said at least one step of mechanicaltreatment is chosen among mechanical treatments exerting a shear action,in order to delaminate said cellulose fibers and to obtain saidnanocelluloses, wherein said oxidative cleavage of said cellulosefibers, catalyzed by said at least one LPMO, facilitates thedelamination of these cellulose fibers during said at least one step ofmechanical treatment.
 2. The process for producing nanocelluloses asclaimed in claim 1, wherein the LPMOs are chosen from the enzymescapable of carrying out a cleavage of the cellulose by oxidation of atleast one of the carbon atoms in positions C₁, C₄ and C₆ of the glucosering.
 3. The process for producing nanocelluloses as claimed in claim 2,wherein the LPMOs are chosen from the AA9 and AA10 families of the CAZyclassification.
 4. The process for producing nanocelluloses as claimedin claim 1, wherein the LMPOs are chosen from the LPMOs derived fromPodospora anserina.
 5. The process for producing nanocelluloses asclaimed in claim 4, wherein the LMPOs are chosen from PaLPMO9A (GenbankCAP68375), PaLPMO9B (Genbank CAP73254), PaLPMO9D (Genbank CAP66744),PaLPMO9E (Genbank CAP67740), PaLPMO9F (Genbank CAP71839), PaLPMO9G(Genbank CAP73072) and PaLPMO9H (Genbank CAP61476).
 6. The process forproducing nanocelluloses as claimed in claim 1, wherein the electrondonor is chosen from ascorbate, gallate, catechol, reduced glutathione,lignin fragments and fungal carbohydrate dehydrogenases.
 7. The processfor producing nanocelluloses as claimed in claim 1, wherein thecellulose-based substrate is obtained from wood, a cellulose-richfibrous plant, beetroot, citrus fruits, annual straw plants, marineanimals, algae, fungi or bacteria.
 8. The process for producingnanocelluloses as claimed in claim 1, wherein the cellulose-basedsubstrate is chosen from chemical papermaking pulps.
 9. The process forproducing nanocelluloses as claimed in claim 8, wherein thecellulose-based substrate is chosen from chemical wood papermakingpulps.
 10. The process for producing nanocelluloses as claimed in claim9, wherein the cellulose-based substrate is chosen from at least one ofthe following chemical wood papermaking pulps: bleached pulps,semi-bleached pulps, raw pulps, bisulfite pulps, sulfate pulps, sodiumhydroxide pulps, kraft pulps.
 11. The process for producingnanocelluloses as claimed in claim 1, wherein, following said at leastone step of mechanical treatment, said process comprises apost-treatment step.
 12. The process for producing nanocelluloses asclaimed in claim 11, said wherein the process comprises a post-treatmentstep chosen from an acid treatment, an enzymatic treatment, anoxidation, an acetylation, a silylation, or else a derivatization ofcertain chemical groups borne by the nanocelluloses.
 13. The process forproducing nanocelluloses as claimed in claim 1, wherein thenanocelluloses obtained consist of cellulose nanofibrils and/or ofcellulose nanocrystals.
 14. The process for producing nanocelluloses asclaimed in claim 1, wherein said at least one step of mechanicaltreatment, exerting a shear action, comprises at least a homogenizationtreatment.
 15. The process for producing nanocelluloses as claimed inclaim 1, wherein said at least one step of mechanical treatment,exerting a shear action, comprises at least a microfluidizationtreatment.
 16. The process for producing nanocelluloses as claimed inclaim 1, wherein said at least one step of mechanical treatment,exerting a shear action, comprises at least an abrasion treatment. 17.The process for producing nanocelluloses as claimed in claim 1, whereinsaid at least one step of mechanical treatment, exerting a shear action,comprises at least a cryomilling treatment.